Subunit structure and ion transport mechanism of vacuolar ATPase

(i)我们成功地对肠球菌V-ATP酶进行了大规模的纯化，从20升培养物中获得了约30 mg的纯化酶，该酶包含9个亚基，A，B，C，D，E，F，G，I，K。SDS-PAGE显示，V1亚基与EDTA孵育后形成V0 V1复合物，V0部分由I和K亚基组成，可释放出A、B、C、D、E、F和G七个亚基。(ii)肠杆菌V-ATP酶催化Na+或Li+的转运，并与ATP水解偶联。这表明该多亚基酶的NtpK蛋白脂亚基的谷氨酸残基（Glu 139）是这些离子转运的结合位点。我们建立了一个互补系统，从ntpK基因与其缺失突变体，并发现ATP酶活性消失后，取代谷氨酸139天冬氨酸。因此，NtpK的酸性残基的侧链长度对于该ATP酶反应是重要的。(iii)我们研究了离子结合步骤，以E. hiagenia Na+-转位ATP酶。Na+与纯化的V-ATP酶结合的动力学表明，6个Na+结合/酶分子，具有单一的高亲和力（Kd（Na+）=15 μ M）。阳离子结合位点的数量与V-ATP酶蛋白脂形成由六聚体组成的转子环的模型一致，每个六聚体具有一个阳离子结合位点。这表明Na ~+-ATPase的Na ~+结合位点很容易从水相进入。

(i) We succeeded in the large-scale purification of Enterococcus hirae V-ATPase achieving column chromatographies ; about 30 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, K, was obtained from 20 liter culture. SDS-PAGE revealed that seven subunits, A, B, C, D, E, F and G, are releasable as the V1 subunit form thew V0V1 complex by incubation with EDTA ; the V0 portion consists of I and K subunits.(ii) E.hirae V-ATPase catalyzes translocation of Na+ or Li+ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) of NtpK proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. We established a complementation system from the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 to aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.(iii) We investigated the ion binding step to E.hirae Na+-translocating ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 Na+ bound/enzyme molecule, with a single high affinity (Kd (Na+) =15 microM.The number of cation binding sites is consisitent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. This suggest that the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase.